Working with 3D scaffolds poses some in vitro analysis challenges, including transitioning current protocols to the new format seamlessly. XanoMatrixTM scaffolds have been designed with this in mind. Popular assays for cell growth and metabolism such as AlamarBlue and MTT can be used to assess cell function and proliferation on the scaffolds just as you would for cells grown on tissue culture plastic. No transfer of the scaffold or large volumes of reagent are necessary.
The following is an example of a simple cell growth assay of XanoMatrix scaffolds compared to tissue culture plastic as a standard using AlamarBlue (Invitrogen). Refer to the Protocols section for detailed methods.
MDA-MB-231 Cancer Cell Line
The MDA-MB-231 human breast cancer cell line displays an invasive phenotype, and has been shown to produce experimental metastasis in mice, and as such is an ideal candidate for investigating cell proliferation in a 3D environment.
MDA-MB-231 cells were seeded onto 48-well XanoMatrixTM plates or on tissure culture (TC) treated plates at a density of 5000/cm2 and grown over time in DMEM-high glucose with 10% FBS at 5% CO2. The AlamarBlue assay was performed as per the manufacturer instructions by removing the growth media from the cells and adding fresh media with 10% AlamarBlue reagent. A standard of known cell densities was also generated previously to normalize fluorescent readings from the plate reader to total cell numbers. After incubation in the reagent, 100 ul of the media was transferred to a 96-well plate and fluorescence was measured at 546/590 nm using a Fluostar plate reader.
Over 9 days, the TC-treated plate showed a high amount of growth initially, then fell off as the cells became overconfluent and started peeling off the well. However, cells grown on XanoMatrixTM showed steady, consistent growth that continued past 2 weeks growth (not shown).
MDA-MB-231 grown on XanoMatrixTM scaffolds or TC-treated plates were also subjected to increasing levels of chemotherapeutic drugs paclitaxel and vincristine sulfate to assess the sensitivity to these drugs. Cells were grown for 2 days then media exchanged for varying concentrations of drugs for 24 hours. AlamarBlue assay was then done to count living cells.